Statement of the Challenge Area and the specific Challenge Topic Research Area: This application addresses Broad Challenge Area (6): Enabling Technologies and Specific Challenge Topic 06-OD-105: Identification of chemical modulators of epigenetic regulators. Project Summary/Abstract: Multicellular organisms have evolved elaborate mechanisms to enable differential and cell-type specific expression of genes. Epigenetics refers to these heritable changes in how the genome is accessed in different cell-types and during development and differentiation. This capability permits specialization of function between cells even though each cell contains the same genome. Over the last decade, the cellular machinery that creates these heritable changes has been the subject of intense scientific investigation as there is no area of biology or indeed, human health where epigenetics may not play a fundamental role.[1] The template upon which the epigenome is written is chromatin - the complex of histone proteins, RNA and DNA that efficiently package the genome in an appropriately accessible state within each cell. The state of chromatin, and therefore access to the genetic code, is largely regulated by specific chemical modifications to histone proteins and DNA, and the recognition of these marks by other proteins and protein complexes. The most important modifications of histones and DNA include: histone lysine and arginine methylation;lysine acetylation;DNA cytosine methylation;and histone sumoylation, ubiquitination, ADP-ribosylation and phosphorylation.[2] Many of these modifications create a binding site to recruit other proteins which can 'read'these marks and lead to cell-type and environmentally appropriate gene expression or repression. Given the wide-spread importance of chromatin regulation to cell biology, the enzymes which produce these modifications (the 'writers'), the proteins that recognize them (the 'readers'), and the enzymes that remove them (the 'erasers') are critical targets for manipulation in order to further understand the histone code and its role in biology and human disease. Indeed, small molecule inhibitors of histone de-acetylases have already proven useful in the treatment of cancer.[3] A systematic and therapeutically unbiased approach to further development of chemical probes for the writers, readers and erasers of the histone code is a major challenge and opportunity for the biomedical community. Cell penetrant, small molecule chemical probes that modulate the regulation of chromatin state are of great significance in the fields of epigenetics, oncology, developmental biology, neurology, stem cell fate and regenerative medicine.[3-5] The creation of a 'tool-kit'of potent, selective, well-characterized and cell-penetrant small molecule probes of chromatin regulation will permit biological hypotheses concerning chromatin-state to be tested with confidence in cell-based and animal models of human biology and disease. Given the emerging evidence of dynamic changes in the histone code, small molecule tools will be uniquely useful in assessing this biology in authentic, functional assays. Indeed, the Structural Genomics Consortium (SGC) has recently announced their intention to focus heavily on epigenetics and creation of chemical probes and we will be collaborating with them in this endeavor. The malignant brain tumor (MBT) repeat is a structural domain of ca. 100 amino acids and occurs in 11 human proteins which recognize mono- and dimethyl-lysine modifications of histones.[6] There are no known small molecule binders of MBT domains. This proposal specifically aims to develop potent antagonists of methyl-lysine recognition by human and Drosophila MBT domain containing proteins in order to permit exploration of the biological consequences of blocking this recognition in cell-based and in vivo models with relevance to normal and disease biology. Current understanding of the biological consequences of MBT domain antagonism would suggest that antagonists may be useful in de-differentiation, re-expression of silenced genes and cellular reprogramming.[7-9] Inclusion of Drosophila MBT domains will enable functional studies in this important model system.[8] In order to discover and characterize high-quality probes for MBT domains: assays will be developed for all human and Drosophila MBT domain containing proteins;small molecule hits will be generated by focused screening, virtual screening and structure based design;hits will be optimized for potency, selectivity and cellular activity consistent with their in vitro profile. The probes developed in the course of this research would be made freely available to the academic biology community with no restrictions on use or intellectual property constraints. Funding from this grant will enable the continued employment of two postdoctoral researchers who are currently funded by start-up funds which will expire in 2010. PUBLIC HEALTH RELEVANCE: This proposal aims to develop small, drug-like molecules targeted to proteins that regulate how the DNA code is accessed and utilized in the different cell-types of the body. This is a novel area for exploration with many potential applications in the fields of epigenetics, oncology, developmental biology, neurology, stem cell fate and regenerative medicine. The chemical probes designed, synthesized and validated in this proposal will have applications in the discovery of molecular targets to treat diseases such as cancer and in the development of safe stem cell based therapeutics.